This invention relates to liquid chromatographic systems and more particularly to systems as used in either isocratic or gradient elution of a liquid carrier serving as a mobile phase together with a sample transported thereby through a stationary phase. Generally, the stationary phase is mounted as a packed or open tubular column within an elongate container one end of which is provided with a sample injector and is connected at that end to the output of a pump for delivering the carrier under high pressure. In current systems, the carrier is delivered at pressures which may be as high as in the range of 3,000 to 7,000 psi and even greater.
The carrier phase in isocratic work comprises a fixed proportion of components, for example, 50% water and 50% methanol, while the carrier phase in gradient elution work is programmed to vary between desired values of proportions, i.e., from 90% water-10% methanol, up to 10% water and 90% methanol, over a predetermined interval of time. The central problem of the methods and apparatus for high pressure liquid chromatography (HPLC) work from the point of view of the analytical chemist requires that the system supply a constant flow rate through the column of a known isocratic composition or proportion or known gradient elution composition according to predetermined settings.
It is a primary object of the present invention to provide a liquid chromatographic system which is reliable in both isocratic or gradient proportion operation to provide a known, predetermined composition of the various components of the carrier in accordance with the preset demand.
The problem which must be overcome includes, for example, the actual compressibility of carrier liquid components, the nonlinear variation of their compressibility as a function of liquid component mixtures, the compressibility of pump components including seals, walls, pistons, valves and the like. Reference is made to an article by Klaus Keck, entitled "A New Gradient Mixer for Column Chromatography", Analytical Biochemistry, 39, pages 288 to 296 (1971), showing a typical system for time share mixing of isocratic and gradient carrier mixtures of liquids using switched solenoid valves which are programmed in accordance with various on-off states by a program card and therein showing the mixing occured at atmospheric pressure. In Keck, the mixed solution thereafter is applied to a mixing tank before being drawn off by a suitable pump for insertion into the column. The details of reciprocating pumps and associated components, however, when operated at high pressures causes variations in the amount of each liquid drawn depending upon its location within the fill cycle or stroke. In a typical low pressure solvent mixing system, a reciprocating piston pump is fitted with inlet and outlet check valves, the inlet valve being directed to a take-up carrier source from a storage facility for the same and a programmed valving means which operates at atmospheric pressure through the inlet check valve. On the pump stroke, the pump delivers a charge of the mixture taken up at the indicated high pressure through the output check valve to the column. Thus, if filling with a multi-component mixture A, B, and C, wherein A is taken first, after which the valve switches to B and C, the fill stroke begins by decompression of all the parts as well as the residual or dead volume of the entire pump and associated components, from the high pump stroke pressure to atmospheric fill pressure. By judicious, careful and expensive construction, this decompression volume and the effect thereof can be minimized. However, it is necessary, in any event, to provide piston end clearance, piston and seal tolerances and check valve and connector particulars, all of which contribute to residual volume, as well as the literal dimensional expansion of the pump cylinder walls and compression seals and the like, all of which contribute unknown variables in the time lag between the start of the fill stroke at the beginning of decompression and the actual opening of the inlet check valve to take in the first of the components of the carrier. While it might be thought possible to measure inlet check valve opening in some direct way, this has not proved to be practical and the present invention relies on an indirect measurement of inlet check valve opening, which indirect measurement accounts for and provides for compensation for all of the foregoing decompression and residual volume compressibility variables including the compressibility of the carrier solvent mixture.